1. Field of Invention
The invention is in the field of medicine and can be used to assess status or detect connective tissue (and/or organ) pathology by means of clonal analysis of substrate-dependent cells forming the tissue. In particular, the invention relates to aesthetic medicine and can be used for dermal examination and subsequent individual correction of aging and other structural skin changes. The methods of the present invention for evaluation of cell populations (in particular, a fibroblast population) forming connective tissue can be used for examining conditions, pathologies and any changes which are determined, mediated or otherwise related to particularities of regenerative processes occurring in such tissue (and/or organs). The methods are also applicable to prediction of the treatment efficacy, correct changes and pathologies which outcome depends upon regenerative processes, their intensity and duration.
2. Discussion of the Background
A method for analyzing cell aging by the identification of changes in biological parameters of the cells, such as characteristics of cell metabolism under the influence of aging factors, is described in GB2395489 (IPC C12N5/06). This method includes proteomic, genomic, and/or transcriptomic investigations or a combination thereof with the subsequent comparison of the results obtained from such investigations for (i) “young” cells, i.e., cells derived from a young donor, with a few (e.g., 1-5) in vitro culturing passages or cells obtained from a tissue with the minimal exposure to UV radiation, and (ii) “old” cells derived from elderly donors or cells with many (more than 7) in vitro passages or cells from a tissue exposed to high UV radiation, wherein cell cultures are placed in a three-dimensional matrix mimicking biological a tissue (e.g., connective, epithelial, and epidermal tissue). This method is proposed for screening studies for identification of pharmaceutical agents capable of modulation of metabolic processes in aging cells.
Among the drawbacks of this method is its complicated execution and that it requires highly qualified personnel and specialized expensive equipment, which stands in the way of the routine use of the method, especially in practicing esthetic medicine. Furthermore, this method is confined to using only cells in vitro and cannot be used to characterize conditions of a cell population from tissue in vivo, from which such cells are obtained, and, consequently, this method cannot be used for diagnosis of tissues on the cellular level and prognosis of a subsequent treatment method.
A method of determining colony forming units (CFU) of endothelial progenitor cells by characterizing cell preparations for the use in regenerative medicine or clinical transplantology is described in WO 2008110570 (IPC G01N33/50, C12N5/06). This publication describes a method of analyzing influence of substances and conditions of performing the analysis on CFU values of endothelial progenitor cells for the subsequent use as a screening test for identifying a positive or negative influence of individual substances on the proliferation of endothelial progenitor cells. This method involves explanation of the initial cells in a suitable medium with a density preventing contact inhibition of growth of the cells, followed by incubation of the cells for several days and analysis of the formed colonies. This analysis of the colonies was carried out by staining, counting, and ranking colonies by size into two groups. One group included colonies of the cells with the high proliferative potential, wherein the colonies were larger than a particular threshold value, equal to 2-4 mm, depending on a composition of the medium, while the other group included colonies with the normal and low proliferative potential, wherein the size of the colonies was equal to or less than the threshold value.
The drawback of this method is that it could be only used for analyzing endothelial progenitor cells, e.g., human umbilical cord endothelial cells (HUVEC) and human microvascular endothelial cells. Also, this method can be used for studying cells in vitro but cannot be used for characterizing conditions of a cell population of tissue in vivo, from which such cells are derived, and, therefore, it cannot be used for diagnosis of tissue on the cellular level and prognosis of a subsequent treatment method.
The method for analyzing a population of fibroblasts from the skin and muscle tissue of 7-8 week human embryos, which involves trypsinization of the cells, and explanation and incubation in a nutrient medium with the subsequent counting of pleomorphic, spindle- and sail-like cells, is described in RU 2017818 (IPC C12N5/02). This method analyzes postnatal cultures of dermal fibroblasts in regard to their mitotic activity by counting colonies with the certain number and density of the cells.
The drawbacks of this method are its labor intensiveness, the need for special equipment, and subjective and qualitative nature of the obtained results, which makes the interpretation of the results difficult and limits their practical application.
The prognostic method for distraction osteogenesis is proposed in patent RU 2110798 (IPC G01N33/48). The method includes implanting culture of bone marrow cells, for example, isolated from sternum, of an experimental animal or human subject in diffusion chambers into mice. After 7 days, the chambers were withdrawn, and their content was used for cytological preparations in which the number of clusters and monocyte-macrophage and fibroblast colonies were calculated. Based on their ratio, the prognostic index (PI) was calculated, and if PI was >1, abnormalities in bone formation were predicted. The authors declare that the method is low traumatic and allows detecting early term abnormalities in the bone formation which are related to disorders of the immunological regulation of distraction osteogenesis.
The disadvantages of this method include highly specialized orientation (prognosis of distraction osteogenesis based on the number of bone marrow clonogenic cells) which limits the use of the method for studying regenerative processes in other tissues and organs. Moreover, the method requires using a great number of laboratory animals (up to 6 mice per one study), involving, besides ethical problems, the necessity to keep a specialized vivarium and availability of operative qualified personnel that significantly increases the cost and complicate the proposed method.
The prognostic method is also known for surgical treatment of chronic osteomyelitis [SU 1372216, IPC G01N1/28] which includes biopsy, cell isolation and cultivation with subsequent calculation of the grown bone marrow fibroblasts. To increase accuracy of the method, after the biopsy from the osteomyelitis locus, the material is transferred to a media 199 with antibiotics for 12-24 hours, and the cloning efficiency of the bone marrow stromal cells is calculated by the formula: CE=a*100000/n, where: CE=cloning efficiency; a is the number of the grown colonies; n is the number of the bone marrow cells, and when CE values are not greater than 5, they predict the poor outcome of the postoperative period, and when CE values are 5.1-100000, the outcome is favorable.
As it follows from the description, the invention suggests the diagnostic use of only one parameter—cloning efficiency of the cells which are isolated immediately from patient's bone tissue. In particular, in contrast to the method of the present invention, the method of SU 1372216 provides only the analysis of well-defined colonies, without considering their distribution per the cell density and the presence of sub-visible diffuse colonies which may contain essential diagnostic information. The above-mentioned parameter of the cloning efficiency equally depends upon two complementary values, i.e., the number of cells which while cultivated are able to form colonies (so called colony-forming units-fibroblast, CFU-f), and the proliferative activity of such CFU-f (i.e., colony forming rate). Calculation of only well-formed colonies can result in underestimation and, finally, distortion of the results. Moreover, available experimental data show seasonal variations of the proliferative activity of the bone marrow cells which undermine reproducibility of the results that make the patent authors to take additional measures to provide a high proliferative activity of the cells, in particular, to use animal feeder cells which reduce feasibility and increase production cost of the method.